The invention is predicated upon a basic understanding of epithelial cells and tissues studied. Such epithelia, which cover free surfaces and line body cavities and ducts, have been studied microscopically for at least three centuries. Recently the biochemistry and molecular biology of epithelial cells and tissues have been extensively investigated. However, the seemingly simple question of how the cells in epithelial tissues are driven to become specialized has remained unanswered. The present invention provides reagents that allow us for the first time to unravel the inter- and intracellular signals that direct epithelial cell differentiation. More fundamentally, the subject reagents permit one to finally decipher what has been a tangled web of suspected interactions involving a wide variety of cell types, some of them non-epithelial, in order to understand and modulate at a molecular level how the cells are driven to differentiate to fulfill specialized functions in the body. Pertinent background information concerning these heretofore disparate systems follows.
2.1 Abbreviations
By way of introduction, the following abbreviations are used in this disclosure: BPA, bullous pemphigoid antigen; CD3, cellular determinant #3, a lymphocyte surface antigen marker; CP, cicatrical pemphigoid, an autoimmune dermatological disease; EBA, epidermolysis bullosa acquisita, an autoimmune dermatological disease; ECM, extracellular matrix; FAs, focal adhesions; HD-BSA, heat denatured bovine serum albumin; HFK(s), human foreskin keratinocyte(s); HFK-ECM, human foreskin keratinocyte-extracellular matrix; kDa, kilodaltons of molecular mass as determined by SDS-PAGE; MAbs, monoclonal antibodies; Mr, molecular radius by SDS-PAGE, approximating molecular mass; SACs, stable anchoring contacts; and SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
E200, E170, E145, E135, E100, and E36 refer to the constituent and associated glycoproteins of the subject epithelial ligand complex epiligrin, having apparent molecular weights of 200.+-.20 kDa, 170.+-.20 kDa, 145.+-.20 kDa, 135.+-.15 kDa, 100.+-.10 kDa, and 36.+-.5 kDa, respectively.
Ep-1, 1-1, and 8-6 refer to the disclosed cDNA clones deposited under ATCC accession numbers 75540, 75539, and 75538, respectively.
Throughout the specification, the notation "(#)" is used to refer to the documents listed in the appended Citations section.
2.2 Epithelial Cells
The invention is predicated upon a basic understanding of epithelial cells and tissues studied. Such epithelia, which cover free surfaces and line body cavities and ducts, have been studied microscopically for at least three centuries. Recently the biochemistry and molecular biology of epithelial cells and tissues have been extensively investigated. However, the seemingly simple question of how the cells in epithelial tissues are driven to become specialized has remained unanswered. The present invention provides reagents that allow us for the first time to unravel the inter- and intracellular signals that direct epithelial cell differentiation. More fundamentally, the subject reagents permit one to finally decipher what has been a tangled web of suspected interactions involving a wide variety of cell types, some of them nonepithelial, in order to understand and modulate at a molecular level how these cells are driven to differentiate to fulfill their specialized functions in the body. Pertinent background information concerning these heretofore disparate systems follows.
The significance of epithelial tissues as a protective barrier is readily apparent in the body as the lining of body cavities, blood vessels, digestive tract, mammary glands, urogenital, endocrine, reticuloendothelial systems, respiratory surfaces, placenta, and surrounding the nerves and brain. The epithelia also forms the basis for the epidermis, cornea, and conjunctiva.
2.3 Epithelial tissues are rather unique in their ability for continuous regulated self-renewal, and in their ability to polarize and control cellular division and the subsequent differentiation of the daughter cells. In attempting to explain how epithelial cells may decide how and when to differentiate, it has been suggested that perhaps gradients of growth factors or interactions with extracellular matrix (ECM) may influence the cells. However, the biochemical mechanisms remain largely unknown.
2.4 The epithelial basement membrane is a common histological feature of columnar, stratified, and squamous epithelia. Another prominent feature is a proliferative basal (stem) cell layer resting on a basement membrane. When viewed through the light microscope, an epithelial basement membrane may include lucent and dense regions termed, respectively, the Lamina lucida and Lamina densa, which are sandwiched between an overlying cellular stroma (stroma), made up of basal stem cells and fibroblasts, and an underlying collagenous matrix. Basement membranes are thin but continuous sheets that separate epithelium from stroma and surround nerves, muscle fibers, smooth muscle cells, and fat cells (1-4). The molecular composition of the basement membrane varies with specialized cellular functions and with the developmental stages, shape, structure, and architecture of different epithelia (5). In the simplest model, basement membranes contain at least type IV collagen (1, 6-8), laminin (7-8), entactin (9), and heparin sulfate proteoglycans (10-11). When co-electrophoresed in SDS-PAGE (12) under reducing conditions, purified EHS tumor laminin was reported to have apparent molecular sizes of 400 kDa and 200 kDa, entactin was 158 kDa, and nidogen was 100 kDa (Kleinman et al., Biochemistry 25:312-318 (1986)).
2.5 The human skin, for example, is an epithelial tissue composed of the epidermis and the dermis. The dermis is relatively acellular and composed of secreted cell products, e.g., collagens and heparin-sulfate- and chondroitin-sulfate-proteoglycans. In contrast, the epidermis is essentially cellular, containing a layer of cells resting on a basement membrane, termed the basal (stem) cells that are covered by a layer of cornified cells, termed the stratum corneum. Central questions in skin biology have been, (1) how do the cells in the basal layer commit to become cornified, and (2) how do cells decide which daughter cells will become cornified, and which will remain in the basal layer to provide the germinal basis for future generations of cells? Histological examination provides little insight. The viable inner malpighian layers of the skin, from which the cornified cells arise, are composed of the basal cell layer, the stratum spinosum and the stratum granulosum. The cell types in these areas include at least keratinocytes, melanocytes, Merkel cells, Langerhans cells, and migratory immune cells. Cell division in the basal (stem) cell layer forms the basis for the continuous self-renewal of the skin, and it is thought that decisions on the fate of the daughter cells are made in this layer.
2.6 Two types of daughter cells appear to be created by cell division in the basal (stem) cell layers of the skin. The first daughter cell, which will continue to divide; and the second daughter cell, which will differentiate and ultimately become cornified. Distinctive cellular features that may define stages in the differentiation of the second daughter cell include at least the acquisition first, of a flattened cell shape with cytoplasmic keratohyalin granules, ivolucrin, and cytokeratin filaments (characteristics of cells in the stratum spinosum); second, of greater amounts of cytoplasmic keratin and a submembranous envelope formed of proteins cross-linked by epidermal transglutaminase (characteristics of cells in the stratum granulosum); and third, the acquisition of distinguishing features associated with cornified anuclear cells such as extensively cross-linked dense submembranous envelopes (i.e., characteristics of cells in the stratum corneum). The molecular mechanisms determining "first daughter" and "second daughter" status, as well as the mechanisms which control epidermal cell differentiation into cornified anuclear cells, are largely unknown, but these mechanisms appear to be coordinated; i.e., cells enter and leave the malpighian layer at approximately the same rate; they appear to be polarized, i.e., from the basal (stem) cell layer to the apical cornified layers; and they appear to be self-regulating, i.e., processes by which the cornified layers are renewed can effectively compensate for variation in the rate of mechanical sloughing of cells from the surface in different parts of the body. The molecular processes by which this remarkable coordination of cells is achieved in skin or other epithelial tissues are largely unknown, at present.
2.7 The attachment of proliferating, basal (stem) cells to the basement membrane occurs at limited points of cellular contact. Contact of epithelial cells, in general, with the basement membrane has been thought to have potential functional significance for maintaining cellular polarization necessary for asymmetric cell division, e.g., to give rise to the distinctively different types of daughter cells, as well as for sustaining the continuous morphogenetic process through which progeny of stem cells differentiate into cornified epithelial cells in skin or into Schwann cells and cells of the spinous strata surrounding nerves. However, there has been (and is currently) a lack of detailed knowledge regarding the cellular biology and molecular biochemistry involved in these postulated polarization and morphogenetic processes. Thus, the mechanisms controlling proliferation of stem cells and commitment of the daughter cells to differentiation are largely unknown.
2.8 The ultrastructure of the attachment points where basal cells are in contact with the basement membrane exhibits characteristic features that are identifiable in appropriately fixed and stained tissues (and cultures). The ultrastructural features have been termed hemidesmosomes (14-16), focal adhesions (17, 18), and hemidesmosome-like stable anchoring contacts (SACs) (19). Focal adhesions and SAC/hemidesmosomes are structurally and functionally distinct adhesion structures (19, 20). Focal adhesions have been observed in motile cells in association with actin-containing stress fibers (20, 21), while SACs appear to be distinguished as a structural component of stationary cells which only form in vitro after cells stop migrating. The function of SACs and focal adhesions is currently not clear, either with respect to their possible role in motility or to other possible roles in the cell biology of the epidermis. However, it has been observed that the lamina densa may be connected to stroma through anchoring fibrils (22), such as those observed in cells which appear to be linked to hemidesmosomes (23-25). SAC/hemidesmosome structures have also been observed to be associated with cytoplasmic intermediate filaments (26, 27) which have a bullous pemphigoid antigen (BPA) identifiable by indirect immunofluorescence.
2.9 Studies of basal cell interactions with basement membranes have been complicated by lack of suitable in vitro model systems as well as by changes occurring in the structure, shape, and composition of basement membranes during development and acquisition of specialized cellular functions (5). There has been a near total lack of in vitro models by which basal (stem) cells might be studied. Keratinocytes are one in vitro epithelial model system. These cells are not basal (stem) cells, but they do represent a major cellular constituent of epidermis. Human keratinocytes have been isolated and cultured from stratified or squamous epithelia in vitro under controlled conditions either using fibroblast feeder layers and conditioned medium (28-30); medium containing at least epidermal growth factor (31); keratinocyte growth medium (KGM) containing at least hydrocortisone, low-calcium, insulin, and insulin-like growth factor-1 (32, 33) serum-free (34, 35) or supplemented MCDB 153 basal nutrient medium (36). One recent study has suggested that 85-90% of keratinocyte clones, derived from growing and cloning normal human skin keratinocytes, may be derived from the basal (stem) cell layer and 10-15% from the suprabasal layers of the epidermis (36). The presumptive "suprabasal" keratinocytes expressed markers of terminal differentiation (i.e., ivolucrin) but still possessed the ability to synthesize DNA. These findings suggested to the investigators that some "suprabasal" keratinocytes may exist in an altered state of "non-terminal" differentiation wherein they are still capable of cell division (36). Others have termed possibly related strains of keratinocytes "nondifferentiating keratinocytes" (37). Ivolucrin is one marker for keratinocyte differentiation in vitro. It is a cytosolic protein of human keratinocytes with a reported apparent Mr of 140 kDa on SDS-PAGE (38); the gene has recently been reportedly cloned (39) and its regulation studied in cells in vitro (40). Ivolucrin is useful as a marker for an early stage in the terminal differentiation of keratinocytes since it is synthesized shortly after keratinocytes leave the basal (stem) cell layer, at a time when cellular enlargement has begun, but before onset of envelope cross-linking (41, 42). Ivolucrin has been reported to have undergone a relatively rapid evolution with the possibility of 3 alleles in monkeys (43, 44). Cytokeratins are a second useful marker for keratinocyte differentiation in vitro. There are at least five cytokeratins which may be expressed by keratinocytes in vitro using Western immunoblot analysis and commercially available monoclonal antibodies AE1 and AE3: these include cytokeratins No. 5 (58 kDa), No. 6 (56 kDa); No. 14/15 (50 kDa); No. 16 (48 kDa); and No. 17 (46 kDa) (45).
Keratinocyte differentiation can be induced in vitro, at least to the extent that the cells change morphology into cells resembling cornified epithelia. This process can be induced in tissue culture with calcium or with ionophores (46, 47). When such keratinocyte differentiation is induced in tissue culture, epidermal transglutaminase can become activated in the cells with coincident development of a cross-linked submembranous protein envelope. During cross-linking, cytosolic ivolucrin becomes associated with the submembranous protein envelope as do two other proteins which are reportedly found in keratinocytes but not in fibroblasts. These two proteins have reported apparent molecular sizes on SDS-PAGE of 210 kDa and 195 kDa (48). In an in vitro reconstituted system it was suggested that addition of ivolucrin promoted cross-linking of proteins (49). Thus, while keratinocytes are useful as an in vitro model for some molecular processes involved in epithelial differentiation, they are not basal (stem) cells and are clearly distinguished from them with at least ivolucrin as a marker. In addition, the past studies of keratinocytes has not approached at a molecular level the possible interactions which may occur between receptors in basal (stem) cells and ligands in the basement membrane.
2.10 Ligands which mediate the binding of basal (stem) cells to the epithelial basement membrane are largely unknown. The known basement membrane components in the lamina lucida layer of the epithelium include at least laminin, nidogen, and heparin sulfate proteoglycan, and in the lamina densa they include types IV and VII collagen (5, 50, 51). The possible cellular receptors which may bind to these ligands include at least the integrin adhesion receptors (for reviews see 52-55).
2.11 Integrins are a family of receptor glycoproteins with two noncovalently associated polypeptide chains of different molecular sizes (the larger termed the a chain and the smaller the b), forming a structure termed a heterodimer. The respective chains have amino acid sequence homology, and the integrins serve a similar function at least as receptors for cellular adhesion to extracellular matrix glycoproteins. Six a chains and at least four b chains have recently been identified, giving at least 24 different theoretical heterodimers which could act as receptors for cellular adhesion. An alignment of the .alpha.6 chain amino acid sequence with the .alpha..sub.3 chain reportedly showed approximately 37% identity (#56). The molecular events and mechanisms governing control of the biosynthesis and assembly of the different a and b chains in different cells and tissues are largely unknown, as is the possible existence of several of the theoretical integrin structures. In T-lymphocytes, as opposed to epithelial cells, the activation of cells with interleukin-2 is correlated with induction of expression of the .alpha..sub.3 .beta..sub.1 integrin on the cell surface (#57).
2.12 Biological functions of integrins in tissues and cells include (1) the possible mediation of the attachment of T- and B-lymphocytes and platelets to basement membrane via integrins .alpha..sub.3 .beta..sub.1, .alpha..sub.2 .beta..sub.1 and .alpha..sub.6 .beta..sub.4 and (2) a possible role in hemostasis and homeostasis for these integrins, the latter by contributing to the maintenance of the structure of the integument and epithelia (#s 19-21; 57, 58).
2.13 Possible associations between integrins and laminin ligands include reports that the .alpha..sub.2 .beta..sub.1 integrin is a collagen receptor in human fibrosarcoma cells (59, 60) with affinity for laminin in some cells (61, 62). Laminin is a disulfide-bonded glycoprotein complex composed of three distinct polypeptide chains. Laminin was first isolated from mouse Engelbreth-Holm-Swarn (Elts) tumor (#7), and the subunits were originally designated as follows: A (400 kDa), B1 (220 kDa), and B2 (210 kDa). However, in light of many recent reports describing multiple isoforms of laminin, the original subunits of EHS laminin (laminin-1) have now been designated as .alpha.1 (400 kDa), .beta.1 (220 kDa), and .gamma.1 (210 kDa) (#125).
.alpha..sub.6 .beta..sub.4 integrin has also been suggested as a laminin receptor in human colon carcinoma cells (63), but it reportedly does not bind to the E8 domain of laminin, a ligand domain of laminin that interacts with .alpha..sub.6 .beta..sub.1 integrin (64). .alpha..sub.3 .beta..sub.1 is reportedly one of the most widely expressed integrins in tissues and in cultured epithelial and non-epithelial cells. It also has been suggested as a possible nonspecific laminin receptor in cells (57, 65). The reports of an association of .alpha..sub.3 .beta..sub.1 with laminin either have not determined the apparent binding affinity of the interaction or have determined the association by assays which permit only a relational comparison, i.e., relatively strong or weak. Laminin is reportedly a poor ligand for adhesion of cultured human foreskin keratinocytes (20, 21). In tissue culture, antibody reactive with .alpha..sub.3 .beta..sub.1 reportedly substantially inhibited adhesion of human foreskin keratinocytes to HFK-extracellular matrix. In contrast, antibody reactive with .alpha..sub.6 .beta..sub.4 had only a minor effect, but when both antibodies were added together, adhesion of HFK to HFK-ECM was reportedly completely inhibited (20) but no ligand was identified. Thus, it is not apparent whether the interactions of .alpha..sub.3 .beta..sub.1, and .alpha..sub.6 .beta..sub.4 with laminin are physiologically meaningful, whether laminin is a ligand, or what the physiologically meaningful ligands for these integrins may be in skin.
2.14 The distribution of the .alpha..sub.6 .beta..sub.4, .alpha..sub.3 .beta..sub.1, and .alpha..sub.2 .beta..sub.1 integrins in tissues is varied. The .alpha..sub.6 .beta..sub.4 form of integrin is limited primarily in epithelial and Schwann cells surrounding myelinated nerves (64) and is down-regulated in differentiated spinal cells (20, 21, 66). SAC/hemidesmosome structures have also been observed to be associated with bullous pemphigoid antigen (27, 67). In contrast, the .alpha..sub.3 .beta..sub.1 and .alpha..sub.2 .beta..sub.1 integrins are widely expressed in tissue and particularly evident in proliferating epithelial cells (20, 21) and in transformed cells and activated lymphoblastoid cells. At the ultrastructural level, the .alpha..sub.6 .beta..sub.4, .alpha..sub.3 .beta..sub.1 and .alpha..sub.2 .beta..sub.1 integrins have been visualized by association with focal adhesions (rather than SACs) and actin-containing stress fibers in motile cells (20, 21). In addition, .alpha..sub.3 .beta..sub.1 (68) and possibly .beta..sub.2 .beta..sub.1, have been implicated in cell-cell adhesion because they have been observed to relocate from areas of cell-substrate contact to areas of cell-cell contact in cells, and because antibodies to the .beta..sub.1 integrin polypeptide inhibit cell-cell contact in cells in vitro (20, 21, 69). In general, .alpha..sub.6 .beta..sub.4, .alpha..sub.3 .beta..sub.1 and .alpha..sub.2 .beta..sub.1, appear only in the proliferating basal cell layer; .alpha..sub.6 .beta..sub.4 appears to be restricted to regions of the stem cell basal plasma membrane (58, 20, 21); .alpha..sub.3 .beta..sub.1 appears on basal lateral and apical regions of the stem cell plasma membrane; and .alpha..sub.2 .beta..sub.1 appears primarily on the apical and lateral regions of the stem cell plasma membrane (59, 20, 21). Thus, while integrins .alpha..sub.3 .beta..sub.1 and .alpha..sub.6 .beta..sub.4 have been recognized as glycoproteins involved in cell-substrate contact in vitro, the available information has created a tangled web which does not permit a determination of which interactions may be physiologically meaningful in vivo.
2.15 Integrins are reported to play a possible role in lymphocyte activation. It has been reported recently that in T-lymphocytes, the interaction of cells containing .alpha..sub.3 .beta..sub.1 integrin with collagen and a second signal such as initiated by binding of antibody to CD3 to the cell surface integrin may trigger cellular activation. Whether such effects may also be triggered by integrins in non-lymphoid cells is not known, at present. It has been reported that a complex substrate composed of a gel formed from purified laminin, type IV collagen, heparin sulfate proteoglycan, entactin and nidogen induced clustering of melanocytes (13), formation of tubular structures by Sertoli cells (70), in vivo growth of neurons (71), and in vitro growth of Schwann cells and liver cells (72). However, it is not known whether the complex substrate induced these effects, or whether the complex substrate favored the growth of a few cells which already possessed these features. Epithelial cells grown with this complex substrate, in general, were reported to assume a much greater polarity than on plastic, collagen, laminin, or fibronectin, but again the molecular basis for this reported change is at present unclear.
2.16 Malignant transformation of normal human keratinocytes in vitro has been reported to impair their ability to differentiate, stratify, and form cornified epithelia in vitro, and these properties were correlated with inability of the cells to synthesize ivolucrin and re-expression of fetal cytokeratins (73). Studies of ivolucrin tissue distribution in cases of skin and lung carcinoma have also suggested that basal cell carcinomas may be negative for ivolucrin with low transglutaminase activity while squamous cell carcinomas may be positive for ivolucrin (74-77). In these respects the transformed keratinocytes and basal cell carcinomas seemed to resemble basal (stem) cells. However, the validity of such interpretations based on ivolucrin has been brought into doubt by the finding that ivolucrin is universally present in both benign acne and keratotic lesions as well as in malignant lesions in skin (78) or cervical tissues (79).
2.17 The mechanisms by which cancer cells of epithelial origin arise are largely unknown, and since these mechanisms are unknown it is difficult to structure treatments to restore normal growth control to malignant cells. Recently it has been reported that malignant human cells may be induced to assume a non-malignant phenotype in vitro by fusion with diploid human keratinocytes. The non-malignant phenotype in the fused cells was reportedly correlated with the continued expression of ivolucrin as a marker of keratinocyte terminal differentiation, i.e., cells which reportedly lost the ability to produce ivolucrin during in vitro culture also reacquired the ability to grow progressively in animals (80).
2.18 Mechanisms involved in psoriasis and autoimmune dermatological diseases are also largely unknown. However, it is reported that epidermal tissues may show decreased transglutaminase activity and premature appearance of ivolucrin in the basal cell layer (81), suggesting a possible premature terminal differentiation of basal (stem) cells in this disease, but not suggesting any mechanisms by which this condition may be caused or corrected. Similarly, bullous pemphigoid (BP), cicatrical pemphigoid (CP), and epidemolysis bullosa acquisita (EBA), are autoimmune dermatological diseases where autoantibodies have been reported (in some patients) that bind to antigens in pathological and normal basement membranes. In BP and CP, using immunoelectron microscopy, immunoreactants have been reported to be in skin, and associated with the lamina lucida (82-86) while, in contrast, EPA immunoreactants reportedly are localized just below the Lamina densa (87). Autoantibodies present in some BP patients' sera also reportedly bound antigens in the Lamina lucida (88, 89) and in EBA they reportedly bound to antigens in the Lamina densa (87, 90). The apparent molecular sizes on SDS-PAGE reported for the BP antigens were 220 kDa and 240 kDa (91) and the EBA were 290 kDa and 145 kDa (90). Using suction blisters and split skin techniques to separate the basal layer from the basement membrane, BP antigens (BPA) were reportedly identified in the "roofs" of the blisters and split skin (i.e., associated with the cells and not with the basement membrane) while CP antigens were reportedly located in the "floors" (i.e., associated with the basement membrane) (92, 93). BPA has been associated by immunoelectron microscopy with ultrastructural elements resembling SACs (26, 27). These ultrastructural studies have made the association between the presence of immunoreactants in the Lamina lucida and the antibodies that are present (in some patients) to the antigens presumed to be present on the basal surfaces of the basal (stem) cells in BP, but it is not clear at present what significance these findings may have for understanding these autoimmune diseases. Similarly, it is not clear at present how these findings may relate to the cell biology and biochemistry of normal epithelia.
2.19 Epiligrin is a recently elucidated epithelial basement membrane component that mediates cell adhesion via integrins .alpha..sub.3 .beta..sub.1 and .alpha..sub.6 .beta..sub.4 (113). Epiligrin, a complex of several glycoproteins, is located in the lamina lucida of the basement membrane where the complex comes in direct contact with the overlying epithelial cells. A major constituent of this complex is a 170 kDa protein (EI70) that is encoded by the LamA3 gene (#135). E170 is the .alpha..sub.3 chain of epiligrin, not to be confused with the .alpha..sub.3 chain of integrin (#135).
Epiligrin and nicein, a similar glycoprotein complex, have been shown to be absent from the basement membrane of patients with the gravis form of junctional epidermolysis bullosa (#114, 121, 126, 135). Junctional epidermolysis bullosa is a blistering disorder of the skin that is characterized by a separation of basal cells from the basement membrane due to a decreased number of hemidesmosomes (#126-128). These data establish that epiligrin interactions with integrin .alpha..sub.6 .beta..sub.4 in hemidesmosomes are important for anchorage of basal cells to the basement membrane. Furthermore, it was shown that epidermotrophic T-lymphocytes can interact with epiligrin via integrin .alpha..sub.3 .beta..sub.1 and this interaction may mediate T cell infiltration of the epidermis during pathogenic cutaneous inflammation (#123). Taken together, these results indicated that epiligrin interactions with both integrin .alpha..sub.3 .beta..sub.1 and .alpha..sub.6 .beta..sub.4 are physiologically important. Similar observations were made in studies by Weitzman et al. (1993), Niessen et al. (1994), and Rousselle and Aumailley (1994) (#s 129-131).
Epiligrin is the major adhesion ligand present in epidermal basement membranes and it has been shown to mediate basal cell adhesion via integrins .alpha..sub.3 .beta..sub.1 in focal adhesions and .alpha..sub.6 .beta..sub.4 in hemidesmosome adhesion structures (113, 133).